This project will address the basic mechanisms of cortical synapse formation. Three specific synapses, two excitatory and one inhibitory, will be studied in organotypic cultures of hippocampal cortex. Brain slices from neonatal rats will be cultured and studied in the living state using time-lapse laser confocal microscopy. Fluorescent stains and ion indicators will be used to visualize cellular structure and activity patterns. Electrophysiological methods will be used to assess and to manipulate synaptic function. Cells examined physiologically will be reidentified after fixation for immunohistochemical and electron microscopic study. The project will begin with a detailed anatomical, immunohistochemical and electrophysiological baseline study of the in vitro development of three synapses: (1) the excitatory mossy fiber synapse onto pyramidal cells in hippocampal area CA3, (2) the excitatory Schaffer collateral fiber synapse onto pyramidal cells in area CA1, and (3) the inhibitory synapse of basket cells onto pyramidal cells in area CA1. The cultured slice system will then be used to pursue four main questions about the physiology and dynamics of synapse formation: 1. How do axonal growth cones navigate in a cortical tissue environment? 2. How do axonal growth cones and immature dendrites make the initial contact leading to synapse formation? 3. What is the sequence and timing of events between initial contact and establishment of a functional synapse? 4. How does electrical activity influence the formation and stability of synapses? Several specific hypotheses regarding each of these questions will be tested. The proposed studies should provide insights into abnormalities of neural development including birth defects, learning disorders and mental retardation. They may also help to suggest medical procedures for prevention or reversal of neurological deficits associated with trauma and stroke. Finally, they may elucidate the developmental basis for normal aging effects on nervous system function and also for degenerative conditions such as Parkinson's and Alzheimer's diseases.